Keywords

5.1 Introduction

Drought and salinity stresses occur naturally (Dai 2011), and have been expanding worldwide due to human activities such as deforestations, salt mining (Ghassemi et al. 1995), poor irrigation water (Marcum and Pessarakli 2006), and escalating emissions of greenhouse gases (IPCC 2000). Currently, more than 800 million hectares (ha) of land are affected by salinity (Munns 2005), and about one third of the world’s arable land has experienced yield reduction due to cyclical or unpredictable drought (Chaves and Oliveira 2004), which are causing a great threat to crop production. For example, China, India, and the USA, the world’s three major grain producers and exporters, have been suffering serious water shortages in many major agricultural regions. In China, according to the survey by the Ministry of Water Resources, over 25.67 million ha of farmland was annually affected by drought stress during the 15th 5-year plan, which caused production reduction of 3.5 × 1010 kg and economic losses of more than 230 billion Chinese Yuan (http://mt.china-papers.com/1/?p=185213).

Generally, the co-occurrence of several abiotic stresses, rather than an individual stress condition, is even worse for crop production (Mittler 2006). For example, the combined effects of salinity and drought on yield are more detrimental than the effects of each stress alone, as observed in potato (Levy et al. 2013), wheat (Yousfi et al. 2012), and barley (Yousfi et al. 2010). However, most studies to date have addressed the effects of single stresses on plant (Zhao et al. 2010; Wu et al. 2013), and little is known about the physiological and molecular mechanisms underlying the acclimation of plants to a combination of salinity and drought (Mittler 2006). Recent studies have revealed that the response of plants to a combination of different abiotic stresses is unique and cannot be directly extrapolated from the response of plants to each of the different stresses individually (Rollins et al. 2013; Iyer et al. 2013). Breeding of stress-tolerant crops is the most efficient strategy to maintain yield in stress-prone marginal land. It is thus important to identify genetic resources with high tolerance to abiotic stresses, especially those co-occurring in the field, such as salinity and drought, and to understand its mechanisms.

Barley (H. vulgare L.) is the fourth most important cereal crop in the world in terms of production. For its versatile properties, it has been used for animal feed, human food, and beverage (Koornneef et al. 1997). Barley as a staple food is attracting renewed attention, especially in Asia and northern Africa, because of its nutritional value (Baik and Ullrich 2008). In addition to its agricultural importance, barley is a genetic model for other crops. However, much of the genetic variation for improving abiotic stress tolerance has been lost during the process of domestication, selection, and modern breeding (Zhao et al. 2010). Even more, barley has a wider ecological range than any other cereals and is widespread in temperate, subtropical, and arctic areas, from sea level to heights of more than 4500 m in the Andes and Himalayas (Bothmer et al. 1995). Barley can be grown on soils unsuitable for wheat, and at altitudes unsuitable for wheat or oats. Because of its salt and drought tolerance, barley thrives in nearly every corner of the earth, including extremely dry areas near deserts. Barley is a short-season, early-maturing, diploid, and self-pollinating crop, thus it is also an ideal model plant for genetic study of drought and salinity tolerance (Li et al. 2007). Several papers have summarized research on barley abiotic stress tolerance including drought and salinity tolerance (Zhao et al. 2010; Wu et al. 2013). In this chapter, we review the impact of salinity and drought stress applied singly and in combination in barley through morphological, physiological, biochemical, molecular, cellular, and ultrastructural approaches.

5.2 Drought Stress and Tolerance

Drought is a meteorological term and is commonly defined as a period without significant rainfall or a deficiency of water supply. Generally, drought stress occurs when the available water in the soil is reduced and atmospheric conditions cause continuous loss of water by transpiration or evaporation. Hence, a continuous shortfall in precipitation (meteorological drought) coupled with higher evapotranspiration demand leads to agricultural drought (Mishra and Cherkauer 2010). Agricultural drought is the lack of ample moisture required for normal plant growth and development to complete the life cycle (Manivannan et al. 2008). Although droughts can persist for several years, even a short, intense drought can cause significant damage and harm the local economy. Drought is a worldwide problem, constraining global crop production and quality seriously and recent global climate change has made this situation more serious (Apel and Hirt 2004; Forster et al. 2004; Zhao et al.2010; Budak et al. 2013).

Drought stress is also considered to be a moderate loss of water, which leads to stomatal closure and limitation of gas exchange. Desiccation is a much more extensive loss of water that can potentially lead to gross disruption of metabolism and cell structure and eventually to the cessation of enzyme-catalyzing reactions. Drought is characterized by the reduction of water content, turgor, total water potential, wilting, closure of stomata, and decrease in cell enlargement and growth. Barley is one of the most important cereal crops grown in many developing countries, where it is often subject to extreme drought stress that significantly affects production (Ceccarelli et al. 2007). Investigating the drought-tolerance mechanisms in barley could facilitate a better understanding of the genetic bases of drought tolerance, and facilitate the effective use of genetic and genomic approaches for crop improvement.

5.3 Salinity Stress and Tolerance

Salinity-affected soils are classified into two types: saline and sodic soils. Sometimes, a third type can be categorized as saline-sodic soils. Salt’s negative effects on plant growth have initially been associated with the osmotic stress component caused by decreases in soil water potential and, consequently, restriction of water uptake by roots.

In agriculture, salt stress severely affects the growth and economic yield of many important crops (Maas and Hoffman 1977). Compared with other cereal crops, including wheat, rice, rye, and oat, barley is highly tolerant to salinity, thus offering a means for efficient utilization of saline soil and improvement of productivity in these environments. However, barley still suffers from salt toxicity in many areas of the world. On the other hand, dramatic differences can be found among and within the barley species, providing the potential for developing cultivars with improved salt tolerance. It is predicted that the genetic improvement of salt tolerance will be an important aspect of barley breeding in the future.

5.4 Overlap Between Salinity and Drought Stresses

Salinity and drought stress show a high degree of similarity with respect to physiological, biochemical, molecular, and genetic effects (Sairam and Tyagi 2004). Physiological drought occurs when soluble salt levels in the soil solution are high enough to limit water uptake due to low water potential, thereby inducing drought stress (Lee et al. 2004). The major difference between the low-water-potential environments caused by salinity versus drought is the total amount of water available. During drought, a finite amount of water can be obtained from the soil profile by the plant, causing ever-decreasing soil water potential. In most saline environments, a large amount of water is at a constant, but under low water potential. Plants have a chance to adjust their osmotic potential, which prevent loss of turgor and generate a lower water potential that allows plants to access water in the soil solution for growth (Taiz and Zeiger 2006).

Both stresses lead to cellular dehydration, which causes osmotic stress and removal of water from the cytoplasm into the intracellular space resulting in a reduction of the cytosolic and vacuolar volumes. Early responses to water and salt stress are largely identical except for the ionic component in the cells of plants under salt stress. These similarities include metabolic processes, e.g., a decrease of photosynthesis or increase in the levels of the plant hormonal processes, such as abscisic acid (ABA). High intracellular concentrations of sodium and chloride ions are an additional problem of salinity stress (Bartels and Sunkar 2005). Plants use common pathways and components in response to stresses, a concept known as cross-tolerance, which allows plants to acclimate to a range of different stresses after exposure to one specific stress (Pastori and Foyer 2002; Tuteja et al. 2007). Thus, a salinity-tolerant species could also be drought tolerant or vice versa, and has similar mechanisms to cope with those stresses (Ashraf and O’Leary 1996).

5.5 Mechanisms of Acclimation or Adaptation to Drought and Salinity Stress

Drought and soil salinity are among the most damaging abiotic stresses affecting today’s agriculture. It is understandable that plants are under periodic water stress because of the unpredictable nature of rainfall. Salt stress is often observed in irrigated areas, hydraulic lifting of salty underground water, or spread of seawater in coastal areas. Plants have evolved mechanisms to perceive the incoming stresses and to cope with them by rapid regulation of their physiology and metabolism. Very often, such regulations and responses include feed-forward mechanisms for stress reduction that are in addition to the responses that are seen after stresses have caused irreversible damage to physiological functions. A good example of such a feed-forward mechanism is the ability of plants to regulate their water loss through partial closure of stomata and/or reduced leaf development, long before there is a substantial loss of their leaf turgor or some irreversible damage to inner membrane systems (Zhang et al. 2006a). The physiological responses of plants to survive under water stress include leaf wilting, a reduction in leaf area, leaf abscission, and the stimulation of root growth by directing nutrients to the underground parts of the plants. Besides, the effects of water deficit become more detrimental during reproductive stages of the plant (flowering and seed development), as the translocation of photosynthetic assimilates from leaf to root is reduced which cannot grow more deep in search of water and nutrients. In addition, ABA, the plant stress hormone, induces the closure of leaf stomata, thereby reducing water loss through transpiration, and decreasing the rate of photosynthesis. These responses improve the water-use efficiency of the plant on the short term (Muhammad and Asghar 2012).

5.6 Effects of Drought and Salinity Stress on Plant Morphology and Yield

5.6.1 Growth and Development

Plant responses to drought and salinity are complex and involve adaptive changes and/or deleterious effects. The decrease in the water potential occurring in both abiotic stresses results in reduced cell growth, root growth, and shoot growth and also causes inhibition of cell expansion and reduction in cell wall synthesis (Chaitanya et al. 2003). According to these authors, drought (likely to salinity) affects the regular metabolism of the cell such as carbon-reduction cycle, light reactions, energy charge, and proton pumping and leads to the production of toxic molecules. Literature has affirmed that plant responses to salt and water stress have much in common. For example, according to Munns (2002), salinity brings a decrease in water uptake by plants as the osmotic potential in the root vicinity will become high and a kind of exosmosis may occur. This will slow down the growth rate, along with a suite of metabolic changes identical to those caused by water stress. Ahmed et al. (2013a) observed that barley plants treated with single or combined stress of salinity (S) and drought (D) showed a significant decrease in plant height, shoot, and root dry/fresh weights, with the largest reduction in the combined stress (D + S). Therefore, most mechanisms to tolerate abiotic stresses like drought and salinity are detrimental to plant development (Fig. 5.1).

Fig. 5.1
figure 1

Possible drought and salt stress tolerance mechanisms in barley plants

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5.6.2 Yield

Many yield-determining physiological processes in plants respond to water stress. Yield is a quantitative trait and many physiological processes are involved. For water stress, severity, duration and timing of stress, as well as responses of plants after stress removal, and interaction between stress and other factors are extremely important (Plaut 2003). For instance, water stress applied at preanthesis reduced time to anthesis, while at postanthesis it shortened the grain-filling period in triticale genotypes (Estrada-Campuzano et al. 2008). In barley (H. vulgare), drought stress reduces grain yield by decreasing the number of tillers, spikes, and grains per plant and individual grain weight. Postanthesis drought stress was detrimental to grain yield regardless of the stress severity (Samarah 2005). In maize, water stress reduced yield by delaying silking, thus increasing the anthesis-to-silking interval. This trait was highly correlated with grain yield, specifically ear and kernel number per plant (Cattivelli et al. 2008). Following heading, drought had little effect on the rate of kernel filling in wheat, but its duration (time from fertilization to maturity) was shortened and dry weight reduced at maturity (Wardlaw and Willenbrink 2000).

Crop growth in saline medium is severely affected at different stages of the plant life cycle. It was suggested by Shannon et al. (1994), that overall plant response depends upon the concentration of salts in the tissue, composition of salts, the exposure time, and climatic conditions as well. The commonly observed adverse effects of salinity on Brassica species include the reduction in plant height, yield, as well as deterioration of the quality of the product (Kumar 1995). In barley and wheat, salinity stress lowered grain yield by reducing grain number and individual grain size (Harris et al. 2010). The plasticity of grain number and stability of grain size was found in another study in response to salinity (Sadras 2007). Ahmed et al (2013b) observed that the reduction in spike length was noticeably less in Tibetan wild barley than cultivated barley treated with single or combined stress of salinity and drought. Moreover, the 1000-grain yield and the filled grains per spike measurements were correlated, which may explain the yield loss in cultivated barley compared to Tibetan wild barley under combined drought and salinity during the anthesis stage. The decline in yield decline was possibly associated with the reduction in spikelet fertility and grain filling (Ahmed et al. 2013b).

In summary, prevailing drought and salinity reduce the plant growth and development, increase flower abscission, reduce grain size due to poor grain filling which arises due to the reduction in the partitioning of photosynthetic assimilate, and decrease carbohydrate metabolism.

5.7 Physiological and Biochemical Bases for Drought and Salinity Tolerance in Barley

5.7.1 Plant Water Relations

Leaf water potential, relative water content (RWC), stomatal movements, transpiration, leaf and canopy temperatures are the important characteristics that influence plant water relations. RWC represents plant water status including water uptake by the roots as well as water loss by transpiration, thus reflect the metabolic activity in plant tissue, and hence used as a most meaningful index for water stress tolerance. A decrease in the RWC in response to drought stress has been noted in a wide variety of plants (Nayyar and Gupta 2006). Furthermore, an exposure of plants to drought stress substantially decreased the leaf water potential, RWC, and transpiration rate, with a concomitant increase in leaf temperature as documented in the previous study (Siddique et al. 2001). Although the components of plant water relations are affected by reduced availability of water, stomatal opening and closing are more strongly affected. In barley, the application of the different watering regimes decreased the RWC, midday leaf water potential (ψ w), and leaf osmotic potential (ψ o) (Robredo et al. 2010).

Osmotic effects of salt on plants are due to the lowered soil water potential in the root zone and thus resemble drought stress by affecting the ability of plants to extract water from the soil and to maintain turgor (Sohan et al. 1999). However, at low or moderate salt concentrations (higher soil water potential), plants accumulate solutes and maintain a potential gradient for the influx of water. Under such conditions, Shannon et al. (1984) reported that growth may be moderated, but unlike drought stress, the plant is not water deficient. Several authors found that water potential and osmotic potential of plants became more negative with an increase in salinity, whereas the turgor pressure increased (Meloni et al. 2001; Gulzar et al. 2003). Vysotskaya et al. (2010) reported a similar decrease in leaf water potential with increasing salt concentration in wild barley species (“20–45” and T-1). At 75 mM NaCl, “20–45” plants were characterized by less inhibition of leaf area, root fresh weight, leaf water content, and leaf water potentials than T-1 species and were, therefore, considered more tolerant to salt stress. According to Vysotskaya et al. (2010), these investigators, it was concluded that, under high salt concentration, plants (1) sequester more NaCl in the leaf that lower the osmotic potential and (2) reduce the root hydraulic conductance causing water stress in the leaf tissue. The combined stress of drought and salinity depressed water potential, RWC in cultivated barley, but was unchanged in Tibetan wild barley relative to control (Ahmed et al. 2013a).

5.7.2 Photosynthesis

Photosynthesis, together with cell growth, is among the primary processes to be affected by drought (Chaves 1991) or by salinity (Munns et al. 2006). The effects can be direct, as the decreased CO2 availability caused by diffusion limitations through the stomata and the mesophyll (Flexas et al. 2007) or the alterations of photosynthetic metabolism (Lawlor and Cornic 2002) or they can arise as secondary effects, namely oxidative stress. Anjum et al. (2011) indicated that drought stress in maize led to considerable decline in net photosynthesis, transpiration rate, stomatal conductance, water-use efficiency, intrinsic water-use efficiency, and intercellular CO2 as compared to well-watered control.

Suppression of the photosynthetic capacity by salinity stress has been reported in a number of plant species (Robinson et al. 1983; Ball and Farquhar 1984; Perez-Lopez et al. 2012) and might be due to lower stomatal conductance, depression in specific metabolic processes in carbon uptake, inhibition in photochemical capacity, or a combination of these (Dubey 1997). Tavakkoli et al. (2011) reported specific ion toxicities of Na+ and Cl− reducing the growth of four barley genotypes grown in varying salinity treatments. High Na+, Cl−, and NaCl separately reduced the growth of barley; however, the reductions in growth and photosynthesis were greatest under NaCl stress and were mainly additive of the effects of Na+ and Cl− stress. High concentrations of Na+ reduced photosynthesis mainly by reducing stomatal conductance. Salt-tolerant species, Barque73, had significantly greater photosynthetic rate and water-use efficiency than those of Sahara, Clipper, and Tadmor. It was concluded that high salt tolerance of the Barque73 was associated with a high CO2 assimilation rate, and water-use efficiency.

5.7.3 Chlorophyll Contents

Chlorophyll is one of the major components of photosynthesis, and decrease in chlorophyll content under drought stress has been considered as a peculiar symptom of oxidative stress and may be the result of pigment photooxidation and chlorophyll degradation. Drought stress caused a large decline in chlorophyll a content, chlorophyll b content, and total chlorophyll content in different sunflower varieties (Manivannan et al. 2007). Barley plants grown under drought showed inhibition of chlorophyll synthesis as demonstrated by reduced SPAD (soil-plant analyses development analyses, based on chlorophyll meter readings) values (Zhao et al. 2010). Guo et al. (2009) reported that, after 13 days of drought stress, Martin and HS41-1 (drought tolerant) had much higher chlorophyll contents than Moroc9-75 (drought sensitive).

The chlorophyll contents of leaves decrease in general under salt stress. The oldest leaves start to develop chlorosis and drop-off with prolonged period of salt stress (Hernandez et al. 1995; Gadallah 1999; Agastian et al. 2000). However, Wang and Nil (2000) have reported that chlorophyll content increases under conditions of salinity in Amaranthus. Salinity causes significant decreases in Chl-a, Chl-b, and carotenoid in leaves of barley (Vysotskaya et al. 2010). Ahmed et al. (2013b) reported that barley plants grown under combined drought and salinity treatment showed a marked reduction in chlorophyll content (Chl-a, Chl-b, and carotenoids), accompanied by a sharp decrease in net photosynthesis (Pn), stomatal conductance (gs), and transpiration rate (Tr). These results indicate that photosynthetic inhibition was caused by stomatal factors and by chlorophyll synthesis inhibition.

5.7.4 Chlorophyll Fluorescence

Chlorophyll fluorescence analysis has proven to be a sensitive method for the detection and quantification of changes induced in the photosynthetic apparatus. The chlorophyll fluorescence is based on the measurement of fluorescence signal of dark-adapted plants exposed to continuous light (Govindjee 1995). The dark-adapted samples show characteristic changes in the intensity of chlorophyll fluorescence during the illumination by continuous lights and this effect is called fluorescence induction of Kautsky’s effect. When barley plants are exposed to drought, the values of maximal quantum yield of PSII (Fv/Fm) decrease, which is a reliable sign of photoinhibition (Guo et al. 2009).

Salt stress leads to a decrease in the efficiency of photosynthesis and is known to influence the chlorophyll content and chlorophyll a fluorescence of barley leaves (Fedina et al. 2003). Chlorophyll a fluorescence parameters have been used to study high salt-induced damage to PSII. By measuring 77 K fluorescence emission spectra in dark grown wheat leaves under high salt conditions, it was shown that salt stress inhibits the chlorophyll accumulation by restraining several steps in porphyrin formation (Abdelkader et al. 2008). Delayed fluorescence measurements in Arabidopsis thaliana seedlings have also proved to be useful as a marker for detecting damage caused by salt stress (Zhang et al. 2008). A significant decrease in Fv/Fm by combined drought and salinity (D + S) suggested a possible inhibition of PSII photochemistry, which could be due to insufficient energy transfer from light harvesting chlorophyll complex to the reaction center. Compared with Tibetan wild barley (XZ5), greater decrease in Fv/Fm in cultivated barley (CM72) indicated that PSII of the latter was more sensitive to D + S, suggesting that a higher protective capacity for PSII could be an important tolerance mechanism for barley genotypes (Ahmed et al., 2013a).

5.7.5 Plant Nutrition

Decreasing water availability under drought generally results in limited total nutrient uptake and their diminished tissue concentrations in crop plants. An important effect of water deficit is on the acquisition of nutrients by the root and their transport to shoots (Farooq et al. 2009). In general, moisture stress induces an increase in N, a definitive decline in P and no definitive effects on K (Garg 2003). Influence of drought on plant nutrition may also be related to limited availability of energy for the assimilation of \(\text{NO}_{3}^{-}/\text{NH}_{4}^{+},~\text{PO}_{4}^{3-},~\ \text{and}~~\text{SO}_{4}^{2-}:\) they must be converted in energy-dependent processes before these ions can be used for growth and development of plants (Grossman and Takahashi 2001). As nutrient and water requirements are closely related, fertilizer application is likely to increase the efficiency of crops in utilizing available water. This indicates a significant interaction between soil moisture deficits and nutrient acquisition. It was shown that N and K uptake was hampered under drought stress in cotton (McWilliams 2003). Likewise, P and \(\text{PO}_{4}^{3-}\) contents in the plant tissues diminished under drought, possibly because of lowered \(\text{PO}_{4}^{3-}\) mobility as a result of low moisture availability (Peuke and Rennenberg 2004). In general, drought stress reduces the availability, uptake, translocation, and metabolism of nutrients. A reduced transpiration rate due to water deficit reduces the nutrient absorption and efficiency of their utilization (Farooq et al. 2009).

Salinity hampers the uptake of macro- and micronutrients and the concentrations of sodium (Na+) and chloride (Cl−) in the plant increase, and the concentrations of potassium (K+) and calcium (Ca+) are reduced (Mansour et al. 2005). This together result in inhibition of plant growth due to limitation in the absorption of other ions and nutrients required for growth. It has also been reported that the accumulation of Na+ and Cl− in both cellular and extracellular compartments competes with K+, Ca+, magnesium (Mg2+), and manganese (Mn2+), whereas Cl− restricts the absorption of nitrate, phosphate, and sulfate ions (Termaat and Munns 1986; Romero and Maranon 1994) and ultimately limits plant growth. Further, high levels of salinity may also affect the transport of Cl− and Na+ by inhibiting the specific transport systems of these ions (Maathuis 2006). Ahmed et al. (2013) reported that combined stress (D + S) resulted in higher increase in Ca, Mn, and Fe concentrations in shoots of wild barley (XZ5) than that of cultivated barley (CM72). Concerning root mineral concentrations, drought or salinity stress alone and in combination significantly increased Ca concentrations in both genotypes, while no significant effect on Zn and Cu concentrations was observed. Drought alone and D + S markedly increased Mn concentration in XZ5, but had no effect on CM72 under salinity and D + S treatments. Maintaining higher translocation of Ca, Mn, and Fe maybe an important way to reduce D + S stress or beneficial to improve plant tolerance to drought and salinity stress (Ahmed et al. 2013a).

5.7.6 Oxidative Stress and Enzymatic Regulation

The generation of reactive oxygen species (ROS) is one of the earliest biochemical responses of eukaryotic cells to biotic and abiotic stresses (Apel and Hirt 2004). The production of ROS in plants acts as a secondary messenger to trigger subsequent defense reactions in plants. The most common ROS are hydrogen peroxide (H2O2), superoxide, the hydroxyl radical, and singlet oxygen that formed as a natural by-product of the normal metabolism of oxygen and is crucial in cell signaling. The overproduction of ROS leads to oxidative stress and can cause damage to cellular components.

To minimize the impact of oxidative stress, plants have evolved a complex system of enzymatic antioxidants, superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione reductase (GR), and ascorbate peroxidase (APX), and nonenzymatic antioxidants, ascorbic acid, α-tocopherol, reduced glutathione, β-carotene, Polyamines (PAs), salicylates, compatible solutes such as proline (Pro), glycine betaine (GB), and zeaxanthin that accumulate in higher plants under drought and salinity stress (Ozkur et al. 2009).

Plants enhance the production of antioxidants in order to minimize the detrimental effects of oxidative stress to normalize their metabolic activities under drought- and salinity-induced oxidative stress (Fig. 5.2). Different antioxidants have roles in protecting cells in specific compartments and in particular conditions. It is generally accepted that \(\text{O}_{2}^{-}\) might be converted to H2O2 and then metabolized to water by APX and GR in plants to maintain membrane structures (Foyer and Fletcher 2001). Likewise, several other antioxidant enzyme molecules are responsible to counteract the deleterious effects of ROS. Initially, SOD catalyzes the conversion of \(\text{O}_{2}^{-}\) to H2O2 that is further reduced to water by APX by using ascorbate as an electron donor (Scandalios 2005). Elevated accumulation of antioxidant enzymes such as SOD, CAT, GR, APX, and POD is involved in lowering oxidative injury in caper bush seedlings under drought stress (Ozkur et al. 2009). Yang et al. (2009) reported an increase in the activity of CAT, SOD, POD, APX, and GR at 25 % field capacity as compared with 100 % field capacity. Seckin et al. (2010) observed the opposite patterns in the activities of SOD, CAT, POD, APX, and GR enzymes in response to NaCl stress in H. marinum and H. vulgare. Thus, the antioxidant system of H. marinum functioned at higher rates to suppress an increased ROS formation under salt stress. The significant increase in the activities of SOD, POD, APX, and GR in the NaCl-stressed leaves of H. marinum was highly correlated with the temporal regulation of the constitutive isoenzymes as well as the induction of new isoenzymes. Lower level of lipid peroxidation also revealed a higher free radical-scavenging capacity and protection mechanism of H. marinum against high salinity (300 mM NaCl) than H. Vulgare. Our previous reports (Ahmed et al. 2013b) indicated that CM72 had a higher malondialdehyde (MDA) content than XZ5 not only under D + S treatments but also under drought alone, suggesting less oxidative damage in Tibetan wild barley than cv. CM72. The essential role of antioxidative systems for maintaining a balance between the overproduction of ROS and their scavenging to keep them at appropriate levels for signaling and reinstatement of metabolic homeostasis is well established.

Fig. 5.2
figure 2

Role of antioxidant enzymes in the ROS scavenging mechanism. Exposure to drought and salinity leads to generation of ROS, including singlet oxygen \({{(}^{1}}{{O}_{2}}),\) perhydroxyl radical (H 2 O), superoxide hydroxyl radicals \((O_{2}^{2-}),\) hydroxyl radicals (OH), and hydrogen peroxide (H 2 O 2 ). ROS reactive oxygen species, SOD superoxide dismutase, CAT catalase, POD peroxidase, GR glutathione reductase, APX ascorbate peroxidase, GABAγ -aminobutyric acid, GSH reduced glutathione, MDHAR monodehydroascorbate reductase, DHAR dehydroascorbate reductase, GST glutathione S-transferase

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5.7.7 Compatible Solutes

Compatible solutes are low molecular weight and highly water-soluble compounds that are usually nontoxic even at high cytosolic concentrations. Plants accumulate compatible solutes, such as Pro and GB, sugars in response to drought and salinity to facilitate water uptake (Hare et al. 1998; Ashraf and Foolad 2007). In addition to osmotic adjustments, these osmolytes were suggested to be important for protecting cells against increased levels of ROS accumulation under stress conditions. Major contributors to osmotic adjustment were revealed to be K+ in the early stages of stress and molecules including GB, Pro, and glucose, in the late stress (Nio et al. 2011).

Pro accumulates in the cytosol and the vacuole during stress (McNeil et al. 1999) and was shown to protect plant cells against damages caused by \(^{1}{{\text{O}}_{2}}\) or HO (Matysik et al. 2002). By quenching \(^{1}{{\text{O}}_{2}}\) and directly scavenging HO, Pro might be able to protect proteins, DNA, and membranes (Smirnoff and Cumbes 1989; Matysik et al. 2002). In the recent study, drought stress alone and D + S combined stress caused a marked increase in GB content in XZ5 and XZ16, more so than in CM72 (Ahmed et al. 2013b). Enhanced GB levels in Tibetan wild barley may exert protection on enzyme activity, including enzymes associated with sugar and amino acid metabolism (Chen et al. 2007), leading to greater increases in soluble sugars and Pro in Tibetan wild barley than control. Thus, it is proposed that the two Tibetan wild barley genotypes may acquire more protection than cv. CM72 under stressed environment due to the elevated levels of GB and the greater osmotic protection from higher levels of soluble sugars and Pro.

5.7.8 Plant Secondary Metabolism

Plant produces a large variety of secondary metabolites through several metabolic pathways in normal condition. But different stresses either biotic or abiotic trigger the plant secondary metabolism that results in enhanced production of plant secondary products. Generally, precursors of secondary metabolic pathways are the products of the primary metabolism. To a large extent, secondary metabolites derive from three biosynthetic routes, namely the phenyl propanoid, isoprenoid, and alkaloid pathways. The major source of aromatic secondary metabolites in plants is the phenylpropanoid pathway (Irti and Faoro 2009).

Elevated phenol and flavonoid content were observed under single and combined stresses in the two Tibetan wild genotypes (Ahmed et al. 2014). In salt stressed H. vulgare, significantly higher concentration of flavonoids was observed (Ali and Abbas 2003). The content of protochatechuic acid, caffeic, and chlorogenic acids was increased following drought stress in Matricaria chamomilla (Kováčik et al. 2009). Ahmed et al. (2013c) also observed that the increase of phenolic compounds in the tissue prevented the formation of ROS in Tibetan wild and cultivated barley under combined drought and salinity stresses. In addition, the induced expression of genes related to secondary metabolism (GST, PPO, SKDH, PAL, CAD, and chi2) was demonstrated under all stress conditions in wild barley and accompanied an increase in the activities of the respective enzymes, with the greatest increase observed in XZ5. During rehydration and recovery, the activities of all enzymes increased except for phenylalanine ammonialyase (PAL) and cinnamyl alcohol ­dehydrogenase (CAD), which increased only in XZ5 (Ahmed et al. 2014).

5.7.9 Ultra-Morphology of Plants

Drought and salt stress leads to disintegration of fine structure of chloroplast, instability of the pigment protein complexes, destruction of chlorophylls, and changes in the quantity and composition of carotenoids (Dubey 1997). A wide array of variation has been observed in many studies regarding the effects of salinity stress on chloroplast ultrastructure like swelling of thylakoid membranes of chloroplast in the mesophyll cells of sweet potato leaves (Mitsuya et al. 2000) and also reduced numbers and depth of the grana stacks, and enlargement of starch grains in the chloroplasts of potato (Bruns and Hecht-Buchholz 1990). Hernández et al (1995) observed disorganized thylakoid structure of the chloroplasts, increased number and size of plastoglobuli, and decreased starch content in chloroplasts of plants exposed to drought and salinity stress. Whereas, chloroplasts aggregation, distortion of cell membranes with no signs of grana or thylakoid in chloroplasts were observed in tomato plants exposed to salt stress (Khavari-Nejad and Mostofi 1998). Elevation in the level of NaCl increased swelling of thylakoids and reduced chlorophyll fluorescence in barley seedlings (Zahra et al. 2014). Chloroplasts and mitochondria were affected in a variety-specific manner under all adverse treatments. The organelles of the drought-tolerant wheat cultivar Katya were better preserved than those in the sensitive variety Sadovo. Leaf ultrastructure can be considered as one of the important characteristics in the evaluation of the drought susceptibility of different wheat varieties (Grigorova et al. 2012). The effect of drought and salinity alone and in combination on endosperm starch and protein composition varied with genotypes and treatments. Under drought stress, the endosperm of CM72 grains had smaller starch granules, especially B-type granules, which were located adjacent to crushed cell layer (CCL), while many A-type starch granules in this region were either pitted or showed surface erosion. The appearance of pitting can be associated with degradation of the proteinaceous layer, exposing the starch granule to severe stress. However, XZ5 and XZ16 showed more protein deposited on the surface of starch granules under drought stress (Ahmed et al. 2013c).

5.8 Identification of QTLs Controlling Drought and Salinity Tolerance in Barley

Quantitative trait locus (QTL) mapping is a powerful approach for locating genomic regions controlling complex traits (Gyenis et al. 2007). By linking phenotypic and genotypic data, QTL mapping enables the identification of the action, interaction, numbers and chromosomal locations of loci affecting particular traits (Miles and Wayne 2008). Large numbers of barley mapping populations have been developed to map genes and QTLs controlling agronomic and quality traits (Table 5.1) and have been reviewed by Fox et al. (2003). Several barley populations have been developed to map the QTLs for drought tolerance in both controlled environments and Mediterranean field trials. These included Tadmor x (ER/Apm) RIL population (Teulat et al. 1998), Derkado x B83-12 DH population (Forster et al. 2004), Apex x ISR101-23 (Pillen et al. 2003), and Barke x Hor11508 populations (Talame et al. 2004).

Table 5.1 Enhancing drought and salinity tolerance in barley lines/varieties using marker-assisted selection
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Kalladan et al. (2013) used advanced backcross quantitative trait locus (AB-QTL) analysis of a BC3-doubled haploid population developed between the cultivated parent Brenda (H. vulgare ssp. vulgare) and the wild accession HS584 (H. vulgare ssp. spontaneum) to study the contribution of wild barley in improving various agronomic and seed quality traits under postanthesis drought. QTL analysis indicated that wild barley contributed favorably to most of the traits studied under both control and drought conditions. A total of seven hotspot QTL regions with colocalizing QTL for various traits harbored more than 80 % of the stable QTL detected in their study. For yield and 1000-grain weight and their respective drought-tolerance indices, most of the QTLs were derived from Brenda. On the other hand, for traits like seed length and seed nitrogen content, all the QTLs were contributed by HS584, the parent with higher trait value.

Many QTL studies carried out using wild barley as a donor parent for various traits indicated that it is a potential source for trait improvement (Nevo 1992; Volis et al. 2000; Pillen et al. 2004; Li et al. 2005, 2006; Rostoks et al. 2005; Schmalenbach et al. 2009; Schnaithmann and Pillen 2013). In addition, H. vulgare ssp. spontaneum was also found to possess positive alleles for abiotic stresses such as drought and salt (Talame et al. 2004; Suprunova et al. 2007; Ceccarelli et al. 2007; Lakew et al. 2011, 2013). Major hindrances to the utilization of wild species in crop improvement using conventional breeding are the quantitative nature of most of the agronomic traits and the linkage drag of undesirable genes present in wild species (Wang and Chee 2010). One of the breeding strategies to overcome the problem of linkage drag associated with wild genotypes during breeding programs is AB-QTL analysis, which combines QTL detection with the introduction of favorable alleles into the targeted variety (Tanksley and Nelson 1996). In barley, AB-QTL analysis was first reported by Pillen et al. (2003) using a BC2F2 population developed between the cultivar Apex and the wild accession ISR101-23 for various agronomic and malting quality traits. Some of the other studies for improving drought tolerance in barley include Baum et al. (2003), Ceccarelli et al. (2004), Forster et al. (1997), Grando et al. (2001), and Ivandic et al. (2003).

Wild barley H. spontaneum has been recognized as an important source for drought tolerance. A QTL identified on chromosome 4H from H. spontaneum consistently increased grain yield across six test environments with an average yield increase of 7.7 % (Pillen et al. 2003). Talame et al. (2004) identified two QTLs on chromosomes 2H and 5H with relative yield increase ranging from 12 to 22 % under dry conditions. These QTLs could be used as target chromosome regions for the integration of wild barley genes for yield improvement under drought. Lu et al. (1999) suggested that drought tolerance in wild barley is related to their differing genetic abilities of osmotic adjustment under drought conditions. Thus, further genetic mapping and marker-assisted transfer of the osmotic-adjustment genes harbored in the wild progenitor could improve resistance of cultivated barley grown in water-limited environments.

Traditional QTL mapping or biparental QTL mapping based on a single segregating population derived from two homozygous parental genotypes has been the commonly used approach for genetic dissection of salt tolerance in barley and to identify candidate genes (Mano and Takeda 1997; Xue et al. 2009; Ellis et al. 2002; Witzel et al. 2009). This approach provides valuable information on genomic regions that control quantitative traits but it also has limitations due to poor sampling of the allelic variation present in the barley gene pool for each of the loci affecting salt tolerance, lack of segregation, and poor resolution of this type of mapping QTLs. Mano and Takeda (1997) identified QTLs controlling salt tolerance at germination and the seedling stage in barley by interval mapping analysis using marker information from two doubled haploid (DH) populations derived from the crosses, Steptoe × Morex, and Harrington × TR306. The results revealed that the QTLs for salt tolerance at germination in the DH lines of Steptoe x Morex were located on chromosomes 4H, 6H, and 5H, and in the DH lines of Harrington/TR306 on chromosomes 1H and 5H. In both DH populations, the most effective QTLs were found at different loci on chromosome 5H. Genetic linkage between salt tolerance at germination and ABA response was found from QTL mapping. The QTLs for the most effective ABA response at germination were located very close to those for salt tolerance on chromosome 5H in both crosses. The QTLs for salt tolerance at the seedling stage were located on chromosomes 2H, 1H, 6H, and 5H in the DH lines of Steptoe x Morex, and on chromosome 5H in the DH lines of Harrington x TR306. Their positions were different from those of QTLs controlling salt tolerance at germination, indicating that salt tolerance at germination and at the seedling stage was controlled by different loci.

Long et al. (2013) demonstrated that a spring barley collection of 192 genotypes from a wide geographical range was used to identify QTLs for salt-tolerance traits by means of an association mapping approach using a 1000 single nucleotide polymorphism (SNP) marker set. Linkage disequilibrium (LD) decay was found with marker distances spanning 2–8 cM depending on the methods used to account for population structure and genetic relatedness between genotypes. The association panel showed large variation for traits that were highly heritable under salt stress, including biomass production, chlorophyll content, plant height, tiller number, leaf senescence, shoot Na+, shoot Cl- , and shoot, root Na+/K+ contents. The significant correlations between these traits and salt tolerance (defined as the biomass produced under salt stress relative to the biomass produced under control conditions) indicate that these traits contribute to (components of) salt tolerance. Association mapping was performed using several methods to account for population structure and minimize false-positive associations. This resulted in the identification of a number of genomic regions that strongly influenced salt tolerance and ion homeostasis, with a major QTL controlling salt tolerance on chromosome 6H, and a strong QTL for ion contents on chromosome 4H (Long et al. 2013).

Recently, Li et al. (2013) confirmed that the distribution of meta-QTL (MQTL) was similar to that of the initial QTL. Many of these MQTL were located on chromosomes 2H (drought) and 5H (salinity). It inferred that chromosomes 2H and 5H were important for barley abiotic stress tolerance. As expected from trait correlations, 22.8 % of these MQTL displayed overlapping confidence intervals (CIs). These overlapping regions were mainly on chromosomes 1H, 2H, and 4H. The results indicated that the tolerance to diverse abiotic stresses were associated with each other in barley (Li et al. 2013).

5.9 Molecular Approaches for Improvement of Modern Barley

The high-throughput omics analysis, including transcriptomics, proteomics, and metabolomics, will improve comprehensive understanding of drought and salt stress-induced changes in gene-protein‑metabolite (Urano et al. 2010; Sicher et al. 2012). Transcriptomics and proteomics analysis have been widely used in salt-tolerance studies (Du et al. 2008; Zhang et al. 2012). Currently, metabolomics are developed and applied in understanding multiple physiological processes in plants, in combination with other platforms such as transcript profiling and proteomics. Major approaches currently used in plant metabolomics are metabolic fingerprinting, metabolite profiling, and targeted analysis. Main analysis methods include gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis-mass spectrometry (CE-MS), Fourier transformation cyclotron resonance-mass spectrometry (FT-ICR-MS), and nuclear magnetic resonance (NMR; Nicholson et al. 1999; Shulaev et al. 2008). In recent years, metabolomics analysis is being widely used to investigate abiotic stress tolerance of plants (Shulaev et al. 2008; Oliver et al. 2011). In barley root, the metabolite profiling was analyzed in response to drought (Sicher et al. 2012), and combined stress of high temperature and drought (Rizhsky et al. 2004). Metabolome changes were also reported in cultivated barleys in response to salt stress (Widodo et al. 2009, Wu et al. 2013). In these context, several categories of genes which respond to the stress could be differentiated (Fig. 5.3): genes that encode protective but metabolically inactive polypeptides, such as dehydrins, chaperones (including proteases), genes for metabolic pathways leading to the synthesis of low-molecular osmolytes which increase stress tolerance, radical scavengers, or compounds with both functions, and regulatory proteins such as transcription factors, protein kinases, phospholipase C, or 14-3-3 proteins.

Fig. 5.3
figure 3

Stress tolerance factors produced in adaptive responses of a barley plant to drought and salinity stress. CBF C-repeat binding factor, MYB myeloblastosis oncogenes, LEA late embryogenesis abundant, INA ice nucleation-active protein, MYC v-myc avian myelocytomatosis viral oncogene homolog, bZIP basic leucine zipper, MAPK mitogen-activated protein kinase, MAPKK mitogen-activated protein kinase kinase, HVA1 ABA-inducible protein PHV A1, WRKY c-terminal wrky domain, NAC nascent polypeptide-associated complex protein

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Most of the drought- and salt-tolerance genes belong to large gene families with high-sequence similarity distribute in a genome, which brings difficulty in identifying the specific locus for a specific function. More recently, genomic technologies have provided high-throughput integrated approaches (Bartels and Sunkar 2005) to investigate global gene expression responses not only to drought but also to other abiotic stresses (Chaves et al. 2003). Microarray profiling under drought stress has been carried out in different plant species such as Arabidopsis (Oono et al. 2003), rice (Rabbani et al. 2003), barley (Ozturk et al. 2002; Talame’ et al. 2007), and wheat (Mohammadi et al. 2007). These studies identified differentially expressed transcripts of genes involved in photosynthesis, ABA synthesis and signaling, biosynthesis of osmoprotectants, protein stability and protection, reactive oxygen detoxification, water uptake, and a myriad of transcription factors including several members of the zinc finger, WRKY (c-terminal wrky domain), and bZIP (basic leucine zipper) families. Du et al. (2011) showed that two dehydrin genes might contribute to improved drought and salt tolerance of Tibetan and wild barley. Hv-WRKY38 is a barley gene coding for a WRKY protein, whose expression is involved in cold and drought stress response which was mapped close to the QTL region (Mare et al. 2004). Hv-WRKY38 was early and transiently expressed during exposure to low nonfreezing temperature, in ABA-independent manner. Furthermore, it showed a continuous induction during dehydration and freezing treatments. The aquaporin, dehydrin, C-repeat binding factor (CBF) genes, and Hv-WRKY38 may be putative candidate genes that underlie the QTL effect on salt tolerance. Differentially regulated proteins predominantly had functions not only in photosynthesis but also in detoxification, energy metabolism, and protein biosynthesis. The analysis indicated that de novo protein biosynthesis, protein quality control mediated by chaperones and proteases, and the use of alternative energy resources, i.e., glycolysis, play important roles in adaptation to drought and heat stress (Rollins et al. 2013).

Transcriptional factors (TFs) play important roles in the regulation of gene expression in response to abiotic stresses such as drought and salinity. TFs are powerful targets for genetic engineering of stress tolerance, because overexpression of a single TF can lead to the up-regulation or down-regulation of a wide array of stress response genes. Until now, transcription factors have been the most appealing targets for transgenic barley improvement, due to their role in multiple stress-related pathways. Dehydration-responsive element-binding protein 1 (DREB1)/CBF and DREB2 gene function in ABA-independent gene expression while ABA-responsive element (ABRE)-binding protein (AREB)/ABRE binding factor (ABF) functions in ABA-dependent gene expression. NAC (nascent polypeptide-associated complex protein) and MYB (myeloblastosis oncogenes)/MYC (v-myc avian myelocytomatosis viral oncogene homolog) are involved in abiotic stress-responsive gene expression (Uauy et al. 2006). In another study, a barley LEA protein, HVA1 (ABA-inducible protein PHV A1), was also overexpressed in wheat, and the overexpressors were observed to have better drought tolerance (Bahieldin et al. 2005). Transgenic wheat obtained with Arabidopsis DREB and HVA1 protein overexpression was also shown to produce higher yield in the field under drought conditions, but further studies are required to confirm their performance under different environments (Bahieldin et al. 2005). The transformation of oat and rice with the barley HVA1 gene also improved drought and salt tolerance (Xu et al. 1996; Oraby et al. 2005). It is not unreasonable to predict in the following decades: genetically modified (GM) wheat will be transferred to the fields as a common commercial crop. However, to pace this process, new transgenics methodologies should be developed since the current methods are laborious and time-consuming. In a recent study, drought enhancement of bread wheat was established with the overexpression of barley HVA1, using a novel technique, which combines doubled haploid technology and Agrobacterium-mediated genetic transformation (Chauhan and Khurana 2011). Most of the transformed genes are from model plants such as Arabidopsis and rice or from wheat and barley cultivars. These approaches could be applied to wild relatives whose genes may have stronger effects. This hypothesis awaits experimental confirmation and field testing.

Plant miRNAs are approximately 20–24-nucleotide noncoding RNAs that specifically base pair to and induce the cleavage of target mRNAs or cause translational inhibition (Zhang et al. 2006b; Shukla et al. 2008). They have diverse roles in plant development, such as phase transition, leaf morphogenesis, floral organ identity, developmental timing, and other aspects of plant development (Lu and Huang 2008; Rubio-Somoza and Weigel 2011). To date, numerous miRNAs from diverse plant species have been identified and functionally characterized in plant development as well as stress response to biotic and abiotic environmental factors (Eldem et al. 2013). More than 40 miRNA families in plants have been associated with response to abiotic stress such as salt and drought (Sunkar 2010; Covarrubias and Reyes 2010). For instance, miR167, miR168, miR171, and miR396 were found to be drought-responsive miRNAs in Arabidopsis (Liu et al. 2008). In search of potential miRNAs involved in drought response in barley, some of the miRNAs, such as miR156, miR171, miR166, and miR408, were observed as differentially expressed upon dehydration (Kantar et al. 2011). miR166 is an example of many drought-responsive miRNAs that were previously characterized as crucial for cell development. It posttranscriptionally regulates class-III homeodomain-leucine zipper (HD-Zip III) transcription factors, which were demonstrated to be important for lateral root development, axillary meristem initiation, and leaf polarity (Hawker and Bowman 2004; Boualem et al. 2008). It is likely that differential regulation of miRNAs in different tissues is important for adaptation to stress in plants. For example, four miRNAs displayed tissue-specific regulation during dehydration in barley: miR166 was up-regulated in leaves, but down-regulated in roots; and miR156a, miR171, and miR408 were induced in leaves, but unaltered in roots (Kantar et al. 2011). Studying drought-responsive miRNAs and their target gene expression in individual cell types will provide greater insights into miRNA target networks that operate in a cell- or tissue-specific manner under drought stress. Zhou et al. (2013) reported that the overexpression of miR319 impacts plant development and enhances plant drought and salt tolerance. The miR319-mediated down-regulation of target genes in transgenic plants may have caused changes in various biological processes, including those associated with water retention capacity, leaf wax synthesis, and salt uptake beneficial to plants responding to salinity and water deficiency. The manipulation of miR319 target genes provides novel molecular strategies to genetically engineer crop species for enhanced resistance to environmental stress. An increasing understanding of the role of miRNAs in drought and salinity tolerance will enable the use of miRNA-mediated gene regulation to enhance plant drought and salinity tolerance.

Although tremendous efforts have been applied to breed drought- and ­salt-tolerant barley by conventional and molecular approaches, truly drought and salt-tolerant barley cultivars have not been produced that can go to farmer’s field. The promising drought- and/or salt-tolerant genotypes are still in the laboratory and experimental fields. To overcome this bottleneck from the laboratory to the farmer’s field, breeding programs should target specific environments and pyramid tolerance genes because drought and salt stresses are complex and variable in different environments and in different years.

5.10 Conclusions and Future Perspectives

Crop production under field conditions can be decreased by several abiotic stresses and the studies on multifactor interactions are of greater importance than analyses of only one stress. A combination of drought and salinity stress affects the plants to a larger degree and plant reaction cannot be directly extrapolated from the response of plants to individual effect of these two stresses. In the case of drought tolerance, plants potentiate to maintain the metabolic activities even at lower level of tissue water potential by accumulating intracellular osmoprotectants such as Pro, GB, amino acids, and soluble sugars. Besides, scavenging of ROS by enzymatic and nonenzymatic antioxidants, cell membrane stability, expression of aquaporin, and stress-related proteins such as LEA (late embryogenesis abundant) are also the vital mechanisms of drought and salinity stress tolerance.

Marker-trait associations are being identified by the development of a high density SNP assay platform that provides sufficient marker density for genome-wide scans and LD-led gene identification (Waugh et al. 2009). Projects are aiming to exploit the discriminatory LD observed in landrace and wild barley populations for fine mapping and gene identification (e.g., ExBarDiv: http://pgrc.ipk-gatersleben.de/barleynet/projects_exbardiv.php). Highly significant associations can be identified between genome-wide SNPs and drought and salt tolerances in wild progenitors, landraces, and varieties. These approaches offer the possibility of identifying novel allelic variation that may be of considerable value to future crop improvement (Waugh et al. 2009).

Advances are still needed to efficiently explore the extensive reservoir of drought and salt-tolerant alleles within wild germplasm deciphering: (1) the molecular networks those lost during domestication and modern breeding (Fu and Somers 2009); (2) the high-throughput screening of wild germplasm for drought/salt tolerance and their regulation of fitness components; (3) the molecular basis of chromosomal recombination; and (4) the potential regulatory relationship between coding and noncoding regions. This will increase the availability of sequence information and will encourage new breeding strategies by transferring single and multiple interacting networked loci/QTLs from wild relatives to commercial varieties via marker-assisted selection. The International Triticeae Mapping Initiative and the Barley Genome Sequencing Consortia are serving as platforms for international collaborative projects that will ensure the use of extensive drought- and salt-tolerance gene pools for cereal crop improvement.